Project Summary Genomic imprinting is closely associated with human genetic disorders due to its functional hemizygosity. The Peg3 domain on human chromosome 19q13.4/proximal mouse chromosome 7 is also associated with several imprinting-related genetic disorders, including frequent loss of human PEG3 expression in the patients of glioma, breast and ovarian cancers. Thus, the long-term objective of this project is to understand the mechanisms controlling the imprinting of the Peg3 domain. In the previous funding cycle, we predicted that the Peg3 domain is likely controlled by one potential ICR (Imprinting Control Region), the Peg3-DMR (Differentially Methylated Region). We subsequently demonstrated that deletion of the Peg3-DMR has a global impact on the transcription of the Peg3 domain. Paternal and maternal transmission of this targeted deletion also resulted in decreased and increased growth rates of the mouse, respectively, an imprinting phenotype associated with proximal mouse chromosome 7. We also discovered that this potential ICR has a very unusual tandem array of YY1 binding sites, and that reducing the in vivo levels of YY1 protein during oogenesis resulted in target-specific DNA hypomethylation on the Peg3-DMR. Based on these observations, we hypothesize 1) that the imprinting of the Peg3 domain is controlled by the Peg3-DMR and 2) that this control requires YY1 as a main trans factor for targeting de novo DNA methylation during oogenesis. In the current proposal, we will test these hypotheses with the following three aims. Aim1 will further characterize the ICR roles played by the Peg3-DMR using the mutant mice lacking the Peg3-DMR. Aim2 will dissect the exact contributions of YY1 to the observed ICR roles of the Peg3-DMR by generating another KO allele with mutated YY1 sites. Aim3 will investigate potential mechanisms by which YY1 establishes and maintains the allele-specific DNA methylation of the Peg3-DMR. The results from these experiments will provide valuable insights regarding previously unnoticed roles for YY1 in genomic imprinting as well as human genetic disorders linked to the Peg3 domain. PUBLIC HEALTH RELEVANCE: A small number of mammalian genes (less than 200) are not functionally equal between the two parental alleles due to an unusual dosage control mechanism, genomic imprinting. The current proposal is aiming to characterize regulatory mechanisms of genomic imprinting.